1. Field of the Invention
The present invention relates to the field of cell isolation, including the isolation of cells from peripheral circulation, labeling cells of interest magnetically, and using an automatable apparatus to immobilize and isolate viable labeled cells for further testing and culture.
2. Related Art
Background
Utilizing current technologies, it is possible to count the number of circulating tumor cells present in blood from breast cancer patients and predict disease outcome [1,2]. However, due to the lack of additional information about the population of circulating tumor cells, current methods do not offer insights into directing treatment or developing novel therapies. Current targeted treatments based on breast cancer subtypes, e.g., Her2/neu or estrogen receptor (ER) status, only focus on the subtype of the primary tumor [3,4,5]. Recent studies have shown that a portion of breast cancer metastases have different Her2 and ER status compared to the original primary tumors [6,7,8,9]. By analyzing the circulating tumor cells (CTCs) for their genetic characteristics, one can target treatment not only to the primary tumor, but also to cells that may contribute to and serve as surrogate markers of metastases, thus improving survival in women with metastatic disease.
CTCs can be collected through a relatively non-invasive blood draw. However, isolating, purifying, and characterizing these cells has proven challenging. Several current technologies allow for isolation and counting of circulating tumor cells from patient blood samples. Additionally, the expression of two or three biomarkers can also be assessed. However, while the CTCs are enriched during these protocols, they are often heavily contaminated with blood cells, may not be viable, or the RNA may be severely compromised, making it difficult to reliably measure gene expression or simultaneously measure the expression of large numbers of genes. Described below is a robotic device which allows one to obtain completely purified, living CTCs. In combination with multiplex qRT-PCR, genes from single CTCs isolated from the blood of patients with metastatic breast cancer were analyzed for expression levels.
Thus, current techniques partially purify CTCs from blood, but there is still residual contamination with other blood cells. Some techniques also fix and permeabilize the CTCs, making them unsuitable for downstream microarray analysis or in vitro and in vivo biological studies
CTC biology is still poorly defined because most studies assess only CTC burden. The characterization of CTCs is a nascent field; isolation of CTCs specifically for multigene molecular analyses, in contradistinction to counting, is challenging for multiple biological and technical reasons. The cells are fragile, likely a result of mechanical stresses on CTCs in the blood stream and chemical effects of cytotoxic chemotherapy. Moreover, CTCs are extremely rare. For example, 81% of metastatic breast cancer patients will have less than ten CTCs in a 7.5 cc tube of blood containing about 1010 blood cells. Technical factors that impede CTC gene expression analysis in currently available platforms include cell fixation and permeabilization, immobilization, and, most importantly, blood cell contamination. CTC fixation and permeabilization performed prior to fluorescent labeling of CTCs can structurally modify RNA and impact cell viability. CTC immobilization on substrates such as glass slides, filters, or microposts limit single cell manipulation. Finally, even after enrichment, CTCs may be contaminated by thousands of leukocytes (white blood cells, WBCs) that confound expression analysis, requiring bioinformatic techniques to subtract non-CTC gene expression. Thus, direct simultaneous analysis of many gene targets in single human CTCs has yet to be performed.
Certain commercial technologies use immunomagnetic enrichment. Commercial products include the CellTracks® AutoPrep® System and CellSearch™ Circulating Tumor Cell Kit (Immunicon Corporation, Huntingdon Valley, Pa.), MACS® separation technology (Miltenyi Corporation, Bergisch Gladbach, Germany), and the RoboSep® automated cell separator (StemCell Technologies, Vancouver, Canada). With these techniques, the epithelial cells in the blood are labeled with magnetic particles attached to an antibody targeted to an epithelial cell surface marker, usually EpCAM. The blood is processed and external magnets hold the epithelial cells at the side of the tube, while the other blood cells are diluted and pipetted out or eluted through a column. The remaining epithelial cells are then available for immunocytochemical analysis, again amidst 1000-10,000 WBCs. Because of heavy mononuclear cell contamination (whose nucleic acids or proteins would overwhelm any subsequent molecular analyses of the CTCs), most analyses stain and count cells, or limit characterization to one to two immunostains.